DNA replication is regulated by polymerases and several other proteins that act on DNA in association with the bacterial membrane. These interactions are altered by the peptide antibiotic phleomycin. Phleomycin arrests normal DNA replication by causing discrete breaks in cellular DNA and by releasing the DNA from its association with the bacterial membrane. The synthetic polymers poly dT, poly dG, poly U, and poly G are more sensitive than poly dA, poly dC, poly A, and poly C to degradation by phleomycin. We will measure the sensitivity of poly 1 to phleomycin degradation in order to determine whether the presence or absence of an amino group on the 6 carbon of the poly-nucleotide bases is the factor that makes them susceptible to phleomycin. Although phleomycin arrests DNA synthesis in vivo, it enhances synthesis in toluenized bacterial cells, and in lysates of nuclei from resting, GO stage rabbit lymphocytes. We will determine the nature of the phleomycin-induced changes in DNA that enhance template activity, and we will relate these changes to alterations in the normal DNA-membrane, and DNA-DNA-polymerase associations in nuclear lysates. We will use density gradient separations, and polyacrylamide gel electrophoresis analyses to identify changes in the DNA that enhance template activity, and we will relate these changes to alterations in the normal DNA-membrane, and DNA-DNA-polymerase associations in nuclear lysates. We will use density gradient separations, and polyacrylamide gel electrophoresis analyses to identify changes in the DNA and in the DNA-membrane, and DNA-enzyme complexes. BIBLIOGRAPHIC REFERENCE: Reiter, Harvard and Pei-Ling Hsu, 1975: DNA synthesis in phleomycin-treated lysates of GO lymphocyte nuclei. ICN-UCLA Symp. Molecular & Cellular Biology. Vol. 3, pp.822-829. DNA Synthesis and its Regulation. eds, M. Goulian, P. Hanawalt, and C. F. Fox. W. A. Benjamin Inc., Menlo Park, CA.